Azlactone based thermally crosslinkable polymer coating for controlling cell behavior

ABSTRACT

Random copolymers, crosslinked thin films of the random copolymers and cell culture substrates comprising the crosslinked thin films are provided. Also provided are methods of making and using the copolymers, thin films and substrates. The copolymers are polymerized from glycidyl methacrylate monomers and vinyl azlactone monomers. The crosslinked thin films are substrate independent, in that they need not be covalently bound to a substrate to form a stable film on the substrate surface.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 15/665,831, filed Aug. 1, 2017, which is a divisional of U.S. patentapplication Ser. No. 14/658,402, filed on Mar. 16, 2015, the entirecontents of both of which are incorporated herein by reference.

REFERENCE TO GOVERNMENT RIGHTS

This invention was made with government support under 1306482 awarded bythe National Science Foundation. The government has certain rights inthe invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 13, 2020, isnamed 00300-0246-DIV_SL.txt and is 3,155 bytes in size.

BACKGROUND

Regulation of fundamental stem cell behavior using two-dimensionalsynthetic templates in vitro is of immense importance in regenerativemedicine. In particular, human mesenchymal stem cells (hMSCs) are ofgreat interest due to their ability to undergo multilineagedifferentiation, and derivation from adult tissues. Control over cellbehaviors such as adhesion, proliferation and differentiation is likelyto facilitate increased therapeutic applications of stem cells. However,the inherent complexity of the native extracellular microenvironmentmakes decoupling the cause and effect of particular signaling cues (e.g.proteins, soluble factors, cell-cell interactions) difficult. For a trueunderstanding of the identity and concentration of extracellular matrixfragments (peptides) necessary for even simple stem cell adhesion, themicroenvironment needs to be simplified. Although growing cells on achemically defined surface, where cell-surface interactions are knownand quantifiable, is desired, it is rarely achieved. Cells aretraditionally grown on tissue culture polystyrene (TCPS), which, likemost other materials, undergoes rapid adsorption of proteins inbiological fluid, creating a poorly defined interface for cell studies,where identity, density and orientation of the biomolecules is unknown.Hence, a large amount of research has focused on creating synthetictwo-dimensional templates for chemically defined cell culture andexpansion including self-assembled monolayers (SAMs), hydrogels, polymerbrushes, thin films, and layer by layer films. To better regulatecell-template interactions common synthetic templates employpoly(ethylene glycol) (PEG) to provide a “blank slate” background tocells. PEG, when used in combination with specific peptides on synthetictemplates, can provide a powerful platform for regulating stem cellbehavior.

Polymer coatings are one of the few templates that are compatible with awide range of substrates and have good physical stability. However, thepolymer coating must remain insoluble and not delaminate from theunderlying substrate for the duration of the cell culture. This limitsthe composition of the polymers that can be used.

SUMMARY

Random copolymers, crosslinked thin films of the random copolymers andcell culture substrates comprising the crosslinked thin films areprovided. Also provided are methods of making and using the copolymers,thin films and substrates.

One embodiment of a random copolymer is a random copolymer of glycidylmethacrylate and 4,4-dimethyl-2-vinylazlactone comprising the structure:

wherein x any y represent the mole fractions of the polymerized glycidylmethacrylate and 4,4-dimethyl-2-vinylazlactone monomers; and theglycidyl groups and the azlactone groups are distributed randomly alongthe copolymer backbone. In some embodiments, the random copolymercomprises no greater than about 30 mole percent of additional monomer.

Random copolymers of this type can be formed into coatings on asubstrate to provide a coated substrate comprising: a substrate having asurface; and a film of crosslinked random copolymers on the surface ofthe substrate, the crosslinked random copolymers having backbone chainscomprising polymerized 4,4-dimethyl-2-vinylazlactone monomers andmonomers that provide covalent crosslinks between the backbone chains,the crosslinked random copolymers comprising the structure:

wherein x any y represent the mole fractions of the crosslinked monomerand the 4,4-dimethyl-2-vinylazlactone monomers;

represents a crosslink to another copolymer backbone chain; and theglycidyl groups, the crosslinks, and the azlactone groups aredistributed randomly along the copolymer backbone.

Coated substrates of this type can provide a cell culture substratecomprising: a substrate having a surface; and a film comprisingcrosslinked random copolymers on the surface of the substrate, thecrosslinked random copolymers having backbone chains comprisingpolymerized monomers comprising covalently linked peptide chains andmonomers that provide covalent crosslinks between the backbone chains,the crosslinked random copolymers comprising the structure:

wherein x any y represent the mole fractions of the crosslinked monomersand the monomers comprising covalently linked peptide chains;

represents a crosslink to another copolymer backbone chain; Peprepresents a peptide chain; and the crosslinks and peptide chains aredistributed randomly along the copolymer backbone.

An embodiment of a cell culture substrate can be made by: copolymerizingabout 99 to about 85 mole percent 4,4-dimethyl-2-vinylazlactone monomerand about 1 to about 15 mole percent glycidyl methacrylate monomer toform a random copolymers of 4,4-dimethyl-2-vinylazlactone and glycidylmethacrylate; forming a film of the random copolymers on a surface of asubstrate; crosslinking the glycidyl groups on the random copolymers toform a crosslinked random copolymer film; and reacting at least aportion of the azlactone functionalities on the random copolymers withmolecules comprising a peptide chain to covalently bind the peptidechains to the random copolymers. At least a portion of the azlactonefunctionalities may then be reacted with molecules comprising apolyethylene glycol chain to covalently bind the polyethylene glycolchains to the random copolymers.

Another embodiment of a random copolymer is a random copolymer ofglycidyl methacrylate, 4,4-dimethyl-2-vinylazlactone and polyethyleneglycol methyl ether methacrylate comprising the structure:

wherein x, y and z represent the mole fractions of the polymerizedglycidyl methacrylate, 4,4-dimethyl-2-vinylazlactone and polyethyleneglycol methyl ether methacrylate monomers; n represents the number ofrepeat units in the polyethylene glycol chain; and the glycidyl groups,the azlactone groups and the polyethylene glycol groups are distributedrandomly along the copolymer backbone chain. In some embodiments, therandom copolymer comprises no greater than about 30 mole percent ofadditional monomer.

Random copolymers of this type can be formed into coatings on asubstrate to provide a coated substrate comprising: a substrate having asurface; and a film of crosslinked random copolymers on the surface ofthe substrate, the crosslinked random copolymers having backbone chainscomprising polymerized 4,4-dimethyl-2-vinylazlactone monomers,polyethylene glycol methyl ether methacrylate monomer and monomers thatprovide covalent crosslinks between the backbone chains, the crosslinkedrandom copolymers comprising the structure:

wherein x, y and z represent the mole fractions of the crosslinkedmonomer, the 4,4-dimethyl-2-vinylazlactone monomer and the polyethyleneglycol methyl ether methacrylate monomer; n represents the number ofrepeat units in the polyethylene glycol chain;

represents a crosslink to another copolymer backbone chain; and thecrosslinks, the azlactone groups and the polyethylene glycol groups aredistributed randomly along the copolymer backbone.

Coated substrates of this type can provide a cell culture substratecomprising: a substrate having a surface; and a film comprisingcrosslinked random copolymers on the surface of the substrate, thecrosslinked random copolymers having backbone chains comprisingpolymerized polyethylene glycol methyl ether methacrylate monomers,monomers comprising covalently linked peptide chains, and monomers thatprovide covalent crosslinks between the backbone chains, the crosslinkedrandom copolymers comprising the structure:

wherein wherein x, y and z represent the mole fractions of thecrosslinked monomer, the monomers comprising covalently linked peptidechains and polyethylene glycol methyl ether methacrylate monomers; nrepresents the number of repeat units in the polyethylene glycol chain;

represents a crosslink to another copolymer backbone chain; Peprepresents a peptide chain; and the crosslinks, the peptide chains andthe polyethylene glycol groups are distributed randomly along thecopolymer backbone.

An embodiment of cell culture substrate can be made by: copolymerizingabout 15 to about 35 mole percent 4,4-dimethyl-2-vinylazlactone monomer,about 50 to about 85 mole percent cytophobic polyethyleneglycol-containing monomer, and about 1 to about 15 mole percent glycidylmethacrylate monomer to form random copolymers of the three monomers;forming a film of the random copolymers on a surface of a substrate;crosslinking the glycidyl groups on the random copolymers to form acrosslinked random copolymer film; and reacting at least a portion ofthe azlactone functionalities on the random copolymers with moleculescomprising a peptide chain to covalently bind the peptide chains to therandom copolymers.

Other principal features and advantages of the invention will becomeapparent to those skilled in the art upon review of the followingdrawings, the detailed description, and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Illustrative embodiments of the invention will hereafter be describedwith reference to the accompanying drawings, wherein like numeralsdenote like elements.

FIG. 1 shows the reaction scheme for the formation of a peptide- andpolyethylene glycol-functionalized crosslinked copolymer that ispolymerized from glycidyl methacrylate monomers and4,4-dimethyl-2-vinylazlactone monomers, crosslinked, and then reactedwith polyethylene glycol-containing and peptide-containing molecules.

FIG. 2 shows the reaction scheme for the formation of a peptide- andpolyethylene glycol-functionalized crosslinked terpolymer that ispolymerized from polyethylene glycol methyl ether methacrylate monomers,glycidyl methacrylate monomers and 4,4-dimethyl-2-vinylazlactonemonomers, crosslinked, and then reacted with peptide-containingmolecules.

FIG. 3A shows the formation of copolymer linkages formed by reacting anazlactone-functionalized copolymer with an amine-functionalized peptide(upper scheme), a thiol-functionalized peptide (middle scheme), and ahydroxyl group (lower scheme).

FIG. 3B shows the reaction of the N-terminus of a peptide chain with anazlactone group, followed by rearrangement to form an amide linkage.

FIG. 4. Table. 1: Compositions of random terpolymers of polyethyleneglycol methyl ether methacrylate monomers, glycidyl methacrylatemonomers and 4,4-dimethyl-2-vinylazlactone monomers(PEGMEMA-r-VDM-r-GMA)made in accordance with Example 1.

FIG. 5 shows the PMIRRAS analysis of PEGMEMA-r-VDM-r-GMA on a goldsubstrate before and after crosslinking via the polymerized GMAmonomers.

FIG. 6 shows the PMIRRAS analysis of PEGMEMA-r-VDM-r-GMA on a goldsubstrate after reacting with cRGDfK peptide (SEQ ID NO: 2) at variouspHs.

FIG. 7 shows the PMIRRAS analysis of PEGMEMA-r-VDM-r-GMA on a goldsubstrate before and after reacting with 1.0 M ethanolamine and cRGDfCpeptide (SEQ ID NO: 1).

FIG. 8 shows the results of XPS depth profiling on a crosslinked film ofPEGMEMA-r-VDM-r-GMA prior to coupling with cRGDfK peptide (SEQ ID NO:2).

FIG. 9 shows the results of XPS depth profiling on a crosslinked film ofPEGMEMA-r-VDM-r-GMA after coupling with cRGDfK peptide (SEQ ID NO: 2).

FIG. 10 is an XPS map showing the atomic percent of nitrogen over a 1.5mm² area of a crosslinked film of PEGMEMA-r-VDM-r-GMA after couplingwith cRGDfK peptide (SEQ ID NO: 2).

FIG. 11 is a schematic diagram of a crosslinked PEGMEMA-r-VDM-r-GMA thathas been patterned with spots of bound peptide using an elastomerictemplate as a mask.

FIG. 12 shows the average projected cell area of hMSCs on a cell culturesubstrate comprising covalently linked cRGDfK peptide (SEQ ID NO: 2).

FIG. 13 shows the average projected cell area of hMSCs on a cell culturesubstrate comprising covalently linked cRGDfC peptide (SEQ ID NO: 1).

FIG. 14 shows the shows the average projected cell area of hMSCs on acell culture substrate comprising covalently linked cRGDfC peptide (SEQID NO: 1) without pre-soaking, after soaking in MEM+10% FBS for twoweeks prior to seeding the hMSCs, and after soaking in PBS for 1 weekprior to seeding the hMSCs.

FIG. 15 shows the shows the average projected cell area of hMSCs on acell culture substrate comprising covalently linked cRGDfK peptide (SEQID NO: 2) without pre-soaking, after soaking in MEM+10% FBS for twoweeks prior to seeding the hMSCs, and after soaking in PBS for 1 weekprior to seeding the hMSCs.

DETAILED DESCRIPTION

Random copolymers, crosslinked thin films of the random copolymers andcell culture substrates comprising the crosslinked thin films areprovided. Also provided are methods of making and using the copolymers,thin films and substrates.

The copolymers are polymerized from glycidyl methacrylate monomers andvinyl azlactone monomers. The glycidyl methacrylate is present at lowconcentration and provides the random copolymers with a crosslinkingfunctionality. The vinyl azlactone monomers provide reactive pendantazlactone rings on the random copolymer backbone. The crosslinked thinfilms are substrate independent, in that they need not be covalentlybound to a substrate to form a stable film on the substrate surface. Asa result, the thin films can be applied to a wide variety of organic andinorganic substrates.

The azlactone rings on the random copolymers can be reacted with variousmolecules, including those comprising polypeptides and/or polyethyleneglycols, to render the crosslinked thin films suitable for use as cellculture substrates. These substrates provide a chemically definedsurface with long-term stability under cell culture processingconditions.

One embodiment of the random copolymers comprises a random copolymer of4,4-dimethyl-2-vinylazlactone (VDM) and glycidyl methacrylate (GMA)monomers (VDM-r-GMA). The structures of these monomers and a randomcopolymer polymerized from the monomers is shown in FIG. 1. In thatfigure, x and z represent the mole fraction of each monomer in thecopolymer. Some embodiments of the copolymers consist only ofpolymerized VDM and GMA monomers. However, other embodiments comprisesmall amounts of additional monomers. Typically these additionalmonomers will be present in quantities of no greater than 30 mole %.This includes embodiments in which the additional monomers are presentin quantities of no greater than about 20 mol. %, no greater than about10 mol. %, no greater than about 5 mol. %, no greater than about 1 mol.%, or no greater than about 0.1 mol. %. Hydroxyethyl methacrylate(HEMA), methylmethacrylate (MMA) and poly(vinyl alcohol) (PVA) areexamples of additional monomers that can be polymerized into thecopolymers. The VDM-r-GMA copolymers may be free of polyethylene glycolmonomers polymerized into the copolymer backbone chain. The GMA providesa crosslinking functionality. However, only small quantities of the GMAare needed to provide a stable crosslinked thin film. Therefore, therandom copolymers typically comprise from about 1 to about 15 mole % ofthe GMA monomer and from about 85 to about 99 mole % VMA monomer. Thisincludes random copolymers comprising from about 2 to about 12 mole % ofthe GMA monomer. The ability to utilize the crosslinking GMA monomer insuch low concentrations is advantageous because high concentrations ofGMA can lead to non-specific protein and biomolecule adsorption.

Another embodiment of the random copolymers comprises a random copolymerof a cytophobic polyethylene glycol-containing monomer, VDM and GMAmonomers. The cytophobic polyethylene glycol-containing monomer confersthe copolymers with resistance to the non-specific binding ofbiomolecules and protein fouling. Polyethylene glycol methyl ethermethacrylate (PEGMEMA) is an example of a suitable cytophobicpolyethylene glycol-containing monomer. PEGMEMA derivatives, includinghydroxyl terminated PEGMEMA, are also examples. The structures of thePEGMEMA, VDM and GMA monomers and a random terpolymer(PEGMEMA-r-VDM-r-GMA) polymerized from the monomers is shown in FIG. 2.In that figure, x and y represent the mole fractions of the VDM andPEGMEMA monomers in the copolymer. In the embodiment shown here, thecopolymer comprises about 11 mole percent GMA. Some embodiments of thecopolymers consist only of polymerized VDM, GMA and cytophobicpolyethylene glycol-containing monomers. However, other embodimentscomprise additional monomers. Typically these additional monomers willbe present in quantities of no greater than about 30 mol. %. Thisincludes embodiments in which the additional monomers are present inquantities of no greater than about 20 mol. %, no greater than about 10mol. %, no greater than about 5 mol. %, no greater than about 1 mol. %,or no greater than about 0.1 mol. %. Hydroxyethyl methacrylate (HEMA),methylmethacrylate (MMA) and poly(vinyl alcohol) (PVA) are examples ofadditional monomers that can be polymerized into the copolymers. As inthe VDM-r-GMA copolymers, the GMA in the terpolymers provides acrosslinking functionality and is needed in only small quantities.Therefore, the random copolymers typically comprise from about 1 toabout 15% of the GMA monomer. This includes random copolymers comprisingfrom about 2 to about 12% of the GMA monomer. The relative amounts ofVDM and cytophobic polyethylene glycol-containing monomers in thecopolymers will depend, at least in part, in the desired degree ofcytophobicity. By way of illustration only, some embodiments of therandom copolymers comprise from about 15 to about 60 mole percentpolymerized 4,4-dimethyl-2-vinylazlactone monomer and about 30 to about85 mole percent polymerized cytophobic polyethylene glycol-containingmonomer.

The random copolymers may be copolymerized in solution using a varietyof techniques, including free radical polymerization, atom transferradical polymerization (ATRP), and reversible addition fragmentationchain transfer polymerization (RAFT).

Thin, lightly-crosslinked polymeric films, which can be referred to asmats, are formed by crosslinking the backbone chains of the randomcopolymers via the pendant epoxy functionalities provided by the GMAmonomers. As shown in FIG. 2, the resulting crosslinks comprise thestructure —C(O)O—CH₂—CH(OH)—. In some embodiments of the crosslinkedfilms all, or substantially all, of the GMA monomers are crosslinked.However it is also possible to retain some residual, unreacted epoxyfunctionalities. The crosslinked thin films can be formed on a substrateby applying a solution of the random copolymers onto the surface of thesubstrate and thermally inducing crosslinking reactions to form aninsoluble film that can swell in aqueous media. Many suitable techniquesfor applying a thin layer of the copolymers to a surface are known.These include spin coating and casting techniques, such as doctorblading. Using these techniques, the crosslinked random copolymer filmscan be formed over very large surface areas. In addition, because thethin films are substrate independent, they can be formed as stable filmson many different substrates, including both organic and inorganicsubstrates. For example, the thin films can be formed on polymericsubstrates, such as polycarbonate, polystyrene orpolyethyleneterephthalate substrates. They can also be formed on metal,semiconductor and oxide substrates. Specific examples of these includeglass, gold and silicon substrates. The substrates may take differentforms. For example, the substrate may be a planar or substantiallyplanar substrate, such as might be found in a planar cell culture well.Alternatively, the substrate may be a microcarrier substrate comprising,for example, a plurality of microcarrier beads for use in a suspensionculture.

The thinness of the crosslinked random copolymer films helps to renderthem stable against delamination from the substrate in solution. Thus,the films desirably have a thickness of no greater than about 100 nm(e.g., from about 5 nm to about 100 nm). This includes films having athickness of no greater than about 50 nm, further includes films havinga thickness of no greater than about 30 nm, and still further includesfilms having a thickness of no greater than about 10 nm. The stabilityof the thin films can be measured by their ability to resistdelamination and degradation in an aqueous solution for an extendedperiod of time. For example, some embodiments of the crosslinked randomcopolymer thin films do not delaminate or degrade in deionized water at37° C. for a period of at least 30 days.

Cell culture substrates are formed by covalently bonding peptide chainsalong the copolymer backbone using polymer-peptide linkers. Theselinkers can be formed by reacting at least a portion of the pendantazlactone functionalities with peptide-containing molecules via anucleophilic ring opening of the azlactone. These peptide couplingreactions can take place at room temperature in aqueous media at lowpeptide concentrations and without activation steps. As illustrated inFIG. 2, the resulting amide linkers comprise the structure—C(O)—NH—C(CH₃)₂—C(O)—. Suitable nucleophiles for the ring openingreaction include primary amines and thiols. Therefore, the linkers canbe formed, for example, via reactions between the azlactone group andthe N-terminus of a peptide chain, a lysine side chain or a cysteineside chain.

A schematic diagram showing polymer-peptide linkers is provided in FIG.3. The top panel of FIG. 3A shows a polymer-peptide linker formed byreacting a pendant azlactone functionality with an amine-functionalizedpeptide, while the middle panel shows a polymer-peptide linker formed byreacting a pendant azlactone functionality with a thiol-functionalizedpeptide to form a thiol ester bond. An N-terminal cysteine on thepeptide chain may be particularly useful with the PEGMEMA-r-VDM-r-GMAcopolymers. In these systems, the thiol of the cysteine initially reactswith the azlactone group to form a thioester bond. However, due to theproximity of the N-terminal amine, there is molecular rearrangement, tothe more favorable and stable amide bond. (Conditions for promotingrearrangement include a 15 minute soak at room temperature in PBS pH7-8, after initial coupling.) The reaction of the N-terminus of apeptide chain with an azlactone group, followed by rearrangement to forman amide linkage is shown in FIG. 3B.

The polymer-peptide linkages in the cell culture substrates are stablefor extended periods under cell culture processing conditions. Forexample, the polymer-peptide linkers may be stable against peptidedetachment for a period of at least 2 days in either of, or both of,phosphate buffered saline (PBS) and minimum essential medium, alpha(αMEM)+10% fetal bovine serum (FBS) at 37° C. This includes embodimentsin which the polymer-peptide linkers are stable against peptidedetachment for a period of at least one week in either of, or both of,PBS and αMEM+10% FBS at 37° C., and further includes embodiments inwhich the polymer-peptide linkers are stable against peptide detachmentfor a period of at least two weeks in either of, or both of, PBS andαMEM+10% FBS at 37° C.

A variety of peptides including very large peptides (i.e., proteins) canbe linked to the copolymers, provided they contain a free thiol oramine. For adhesion peptides it may be desirable for the peptides to be:Arg-Gly-Asp-containing peptides (RGD peptides) (cyclic and linear),optionally, with the PHSRN synergy site (SEQ ID NO: 3);Arg-Glu-Asp-Val-containing peptides (SEQ ID NO: 4) (REDV peptides (SEQID NO: 4)); Ile-Lys-Val-Ala-Val-containing (SEQ ID NO: 5) (IKVAVpeptides (SEQ ID NO: 5)), and/ or Tyr-Ile-Gly-Ser-Arg- containingpeptides (SEQ ID NO: 6) (YIGSR peptides (SEQ ID NO: 6)). However, othertypes of peptides can be used, depending on the application. By way ofillustration, for growth factor (GF) sequestering KRTGQYKL peptides (SEQID NO: 7) may be used; for heparin binding TYRSRKY (SEQ ID NO: 8) andTYRKKGLQ peptides (SEQ ID NO: 9) may be used; for BMP-2 receptor bindingEPPSIATSYKLALKTSIVSL peptides (SEQ ID NO: 10) may be used; and for VEGFbinding KLTWQELYQLKYKGI peptides (SEQ ID NO: 11) may be used.

The ability of the cell culture substrates to avoid non-specific bindingof biomolecules and protein fouling, even in serum-containingconditions, can be enhanced by covalently binding cytophobicpolyethylene glycol (PEG) chains to the copolymer backbone usingpolymer-PEG linkers. These linkers can be formed by reacting at least aportion of the pendant azlactone functionalities with PEG-containingmolecules via pegylation—a base catalyzed ring opening hydrolysisreaction. This may be particularly useful for cell culture substratescomprising the VDM-r-GMA copolymers that lack cytophobic polyethyleneglycol-containing monomers. A VDM-r-GMA copolymer having pendant PEG andpeptide chains covalently linked along its backbone is shown in FIG. 1.The mole ratio of covalently linked peptide chains to covalently linkedPEG chain will depend on the desired degree and location of celladhesion on the cell culture substrates. By way of illustration only, insome cell culture substrates the mole ratio of the number of covalentlylinked peptides to the number of covalently linked PEGs will be in therange from about 1:3 to about 0.1:99.9.

The bound peptide chains and/or polyethylene glycol chains may beuniformly distributed on the cell culture substrates, or may bedistributed in a regular or irregular pattern, such that one or moreregions of the substrate are resistant to cell adhesion by virtue of thepresence of cytophobic polyethylene glycol chains, while one or moreother regions (e.g., one or more spots) promote biospecific interactionswith biological cells by virtue of the presence of the covalentlytethered peptides.

The cell culture substrates can be used to culture biological cells byseeding the biological cells onto the cell culture substrate andculturing the seeded cells in an appropriate culture medium underappropriate culturing conditions. Animal stems cells, includingmammalian stem cells, are examples of cells that can be cultured usingthe present substrates. Human stem cells that may be cultured on thecell culture substrate include human mesenchymal stem cells and humanembryonic stem cells. However, the cell culture substrates can be usedto culture other types of adhesion-dependent cells including, but notlimited to, epithelial cells, endothelial cells, epidermal cells,fibroblasts, muscle cells, chondrocytes, osteocytes osteoblasts andadhesion-dependent cancer cells. The cell culture substrates need not beable to promote the growth of the biological cells indefinitely. For thepurposes of this disclosure, a substrate is considered to suitable foruse a cell culture substrate if it is able to promote cell growth for aperiod of at least 20 days, as illustrated in Example 1 below.

EXAMPLES Example 1

This example illustrates a P(PEGMEMA-r-GMA-r-VDM) polymer coating thatincorporates VDM chemistry for use as a template for stem cell growthand expansion.

The formation of both thioester and amide polymer-peptide linkers byring opening of the azlactone is also illustrated. CycloArg-Gly-Asp-Phe-Lys (cRGDfK) (SEQ ID NO: 2) or cyclo Arg-Gly-Asp-Phe-Cys(cRGDfC) (SEQ ID NO: 1) were used as adhesion molecules and hMSC and H1embryonic stem cell attachment was examined. Finally, the applicabilityof these copolymers to coat large area plastic dishes and to passagehMSCs without the use of proteases is demonstrated. Taken together, theresults detailed here, show that the copolymer is useful for large areachemically defined growth and expansion of adult and pluripotent stemcells.

Results and Discussion Design, Synthesis and Coating Formation Process

As a starting point glycidyl methacrylate (GMA) and vinyl azlactone(VDM) were copolymerized to test the copolymerization conditions byliving free radical methods. In fact GMA and VDM could be copolymerizedby atom transfer radical polymerization (ATRP), reversible additionfragmentation chain transfer polymerization (RAFT), as well asconventional free radical polymerization using azobisisobutyronitrile(AIBN), a common initiator. While this copolymer can be used tointroduce both the peptides and PEG chains by ring opening of the VDMwith PEG amine and N terminal of the peptides to impart cell-adhesiveand nonspecific protein adhesion respectively, the quantification of therelative amount of introduced peptide and PEG molecules can bechallenging. Hence, a third comonomer polyethylene glycol methyl ethermethacrylate (PEGMEMA) was introduced to confer nonspecific proteinadhesion and allow more accurate quantification both before and afterformation of the coating. The tercopolymer P(PEGMEMA-r-GMA-r-VDM) wassynthesized by RAFT (FIG. 2) and characterized in solution by protonNMR. The resulting polymer had a molecular weight of over 20 kDa toensure sufficient chain entanglement. Multiple copolymer compositionswith varying amount of VDM but fixed GMA content, were made by RAFT. Thecopolymer compositions are provided in Table 1, which is shown in FIG.4. This example focuses on the copolymer containing 24% azlactone, 11%GMA and the remaining 65% PEGMEMA with a molecular weight of 43,094 Daand dispersity of 1.28. The relatively high molecular weight and lowdispersity indicates that the reaction is well controlled by RAFT. Thecontrolled synthesis of this copolymer also allows characterization ofthe relative amount of VDM and GMA in solution before coating. Thisadvantage distinguished the copolymers from those that are polymerizedor formed on the surface such as layer-by-layer films as well ascommercially available OptiChem® and Synthemax surfaces.

Optimization of Coating Crosslinking

GMA was used to crosslink the thin films. GMA can be annealed at up to210° C. and as low as 70° C., but in general longer times are requiredfor full crosslinking at low temperature. However, crosslinking at lowertemperatures is desirable in order to broaden the applicability of thefilms to plastic substrates, such as polycarbonate and polystyrene cellculture plates. Plastic culture plates and flasks are used extensivelyin general cell culture; therefore the ability to simply coat alreadyexisting products is advantageous and cost effective. Coatings werefabricated by spin coating the copolymer from an ethanol solution, asolvent tolerant of plastic substrates. For optimization of thecrosslinking conditions, silicon was used as the substrate. Crosslinkingwas carried out at temperatures of 160° C., 110° C. and 85° C. The filmwas considered fully crosslinked when the film thickness did not changeafter soaking for an hour in tetrahydrofuran (THF), which is anexcellent solvent for the polymer. Complete crosslinking was achievedwithin 45 minutes at 160° C. The crosslinking was slower at lowertemperatures, taking up to 3 hours at 110° C. and up to 24 hours at 85°C.

Physical Stability of the Coating

During the process of cell growth and expansion, the coatings will beimmersed in aqueous solution for extended timeframes, and therefore thecoating itself should be physically stable. For this system the amountof GMA in the copolymer was 11% to minimize and prevent uncrosslinkedchains (physically entangled) from eluting out over time, causing a slowdecrease in overall thickness. Here the GMA did provide the coatingswith stability, as 92% of the film thickness was retained over a 35 dayperiod while immersed in deionized water at 37° C.

Demonstration of Applicability to a Broad Range of Substrates

Coatings were cast onto gold (Au), silicon, glass, polycarbonate andpolystyrene substrates and thermally crosslinked. The static watercontact angle of the film remained essentially the same (59 degrees)even though the bare substrates have a range of initial hydrophobic andhydrophilic contact angles. This simple test proved the coating can beformed on many types of substrates.

Azlactone Ring Opening Characterized by PMIRRAS

The chemistry for attachment of the peptides to the azlactonefunctionality of the stable crosslinked coatings was optimized. Inoptimizing the chemistry, the goal was to meet the followingrequirements: achieve peptide coupling in aqueous solution forapplicability on polymeric substrates, as organic solvents can severelydegraded the substrate; achieve coupling at low concentrations to reducethe peptide cost for large area samples; and achieve quick coupling(within 1-2 hours), as it is desirable to pattern and analyze multiplepeptides on a given coating. Typically patterning methods are used as acombinatorial screening tool for multiple compositions, and the lowerthe time required for functionalization, the lower chance of leakage andcross talk. The reactivity of the azlactone ring was investigated in thecoatings using infrared spectroscopy, specifically polarizationmodulation-infrared spectroscopy (PMIRRAS), to quantify the reactivityof azlactone ring with a nucleophile (—OH, —SH or —NH₂, FIG. 3).

To evaluate the effect of high temperature (110° C.) crosslinking, ifany, on the azlactone functionality, the crosslinked and uncrosslinkedfilms were compared by PM-IRRAS. FIG. 5 shows that the carbonyl peak at1818 cm⁻¹ from the azlactone ring was still intact after thecrosslinking. The peak located at 1818 cm⁻¹ completely disappeared afteronly one hour reaction at room temperature (FIG. 6), and new peaks at1661 cm⁻¹ and 1525 cm⁻¹ from amide linkages emerged.

Next, the reactivity of the cell adhesive peptide cRGDfK (SEQ ID NO: 2)through the lysine side chain was examined. As a logical starting pointcoupling in phosphate buffered saline (PBS) at pH 7.4 was attempted, asthis is a very common buffer used in cell culture. At neutral pH (7.4)and concentrations of 10 mM or lower, the azlactone ring did not open atroom temperature, even when reacted for 24 hours. It is likely that atlow concentrations of peptide the reaction kinetics is slow and the PEGside chains may pose steric barrier for the deprotonated amine to reactwith the azlactone ring. To facilitate amine coupling at 10 mMconcentration, 1.5 M sodium sulfate was added, and the pH was raised toincrease the amount of bound peptide (FIG. 6). The cRGDfK peptide (SEQID NO: 2) did not react significantly within 1 hour, until the pH wasraised to 9.5, then the peak at 1818 cm⁻¹ completely disappeared (FIG.6). Coupling was confirmed by the emergence of amide peaks at 1661 cm⁻¹and 1535 cm⁻¹. As a control the coating was incubated in pH 9.5 bufferfor 1 hour, in the absence of the amine, and complete hydrolysis of theazlactone ring was observed, leading to the emergence of a carboxylatepeak at 1610 cm⁻¹. This suggests at basic pH there is a competitionbetween amines and hydroxyl groups to open the azlactone ring.

A comparable peptide cyclic cRGDfC (SEQ ID NO: 1) was reacted for 1 hourat 1 mM in PBS at pH 7.4 (FIG. 7). Surprisingly the peak at 1818 cm⁻¹completely disappeared and two very strong amide peaks appeared. Thepolymer peptide linker here was a thioester bond, the two amide bondsappeared due to the peptide backbone. The intensity of the amide peaksshowed that thiols react faster and more completely with azlactones thanamines at neutral pH and at low concentrations.

XPS Analysis of Thin and Thick Coatings

To further probe the reaction of the copolymer coating with cRGDfC (SEQID NO: 1) and cRGDfK (SEQ ID NO: 2), X-ray photoelectron spectroscopy(XPS) was performed. XPS is a common surface characterization tool thatcan be used to determine the surface coverage of small molecules. In ablank coating, elements carbon (C), oxygen (O), and nitrogen (N) werepresent. Each azlactone ring has 1 nitrogen atom (N), which provides amarker to calculate the theoretical maximum peptide concentration. Theconcentration used for solution coupling was decreased from 8.4 mM, 0.84mM, and 0.084 mM and the percent coupled compared for cRGDfK (SEQ ID NO:2) and cRGDfC peptides (SEQ ID NO: 1). The reaction time was kept to 1hour at room temperature. The results show that peptide concentrationsas low as 0.084 mM for cRGDfK (SEQ ID NO: 2), and 0.84 mM for cRGDfC(SEQ ID NO: 1), can be used without adversely affecting the reactionefficiency. In agreement with the PMIRRAS data, the XPS analysis alsosupports more efficient reaction of thiols over amines with azlactone.

XPS typically samples 10 nm into the thickness of the film. Since thecrosslinked coatings are post-functionalized with peptides it isimportant to understand the spatial distribution of the peptides. Tostudy this depth profiling using large Argon clusters with a etch rateof 0.05 nm/sec was used, and XPS data was acquired every 1.5 nm. In XPSdepth profiling, the top layer of the material is continuously removedwhile XPS spectra is being acquired. The azlactone content in anunmodified film was quite uniform throughout the thickness of the layerFIG. 8. The increase in the amount of nitrogen after coupling (FIG. 9)was due to the amide bonds in the peptides. In fact, throughout the 30nm thickness the peptide was relatively well distributed, indicatingthat the peptides diffuse and bind throughout the depth of the coating.

To visualize the localization of the cRGDfK peptide (SEQ ID NO: 2), theatomic percent of nitrogen on the surface of the coating was mapped(FIG. 10) using the XPS mapping function. An elastomeric template (PDMS)was used to create a 1.1 mm diameter spot of the peptide. Elementmapping of N (1 s) atomic percent over the 1.5 mm² area shows the centerof the spot at 3.76 atomic % and the background area at 1.14%. Theatomic percents obtained are in good agreement with the depth profilingstudies and clearly indicate the peptide can bind the coating in a PDMSstencil.

Coatings for Controlled Stem Cell Attachment

After characterization of the coating and optimization of the azlactonepeptide coupling, cell adhesion experiments were preformed to test theeffectiveness of the template as a cell culture substrate. The hMSCadhesion to both cRGDfK (SEQ ID NO: 2) and cRGDfC (SEQ ID NO: 1),passage of hMSCs on the coating, and the stability of thioester versusamide bonds in aqueous environments were investigated.

Stem Cell Attachment to Cyclic Peptides

An elastomeric template was used to pattern multiple peptidecombinations at once into small 1.1 mm diameter spots on the crosslinkedfilm (coating). The patterned structure, shown schematically in FIG. 11,included the plastic (polycarbonate) substrate 1100, the crosslinkedrandom copolymer film 1102, the elastomeric template 1104 defining aplurality of spots 1106, and peptide chains 1108 covalently linked tocrosslinked random copolymer 1102 film in spots 1106. In the 1.1 mmdiameter spots, 1.3 μL of a solution of a mixture of adhesive andnon-adhesive scramble peptide (overall peptide concentration constant at1 mM), was pipetted. This effectively lowered the total amount ofadhesive peptide on the surface while keeping the total peptide contentthe same. Seeding and culture of hMSCs in the presence of 10% fetalbovine serum (FBS) showed an insignificant amount of non-specific celladhesion to the non-adhesive cRADfK (SEQ ID NO: 12) or cRADfC (SEQ IDNO: 13) spots (0% condition) and to the surrounding coating(unmodified). The results showed attachment and spreading of hMSCs onthe spots after 6 hours with increasing amount of adhesive peptide. Toquantify cell attachment, hMSCs were allowed to attach for 20 hours onlarge area samples (1 inch by 0.5 inch) before they were fixed andstained for the actin cytoskeleton. For both the peptides cRGDfK (SEQ IDNO: 2) and cRGDfC (SEQ ID NO: 1) (FIGS. 12 and 13, respectively), thehMSC projected cell area directly correlated with the amount of peptidepresent on the coating, as RGD density increased, the cells increased inprojected area. In general, cells with areas of 800 cm⁻¹ and lower werenot well spread and most likely did not have extended lamellipodia.

Chemically Defined Passage of hMSCs

The ability to passage hMSCs off the coating and reseed them back downonto the same coating could have a large impact on bio manufacturing andthe therapeutic applications of hMSCs. Using Versene solution (LifeTechnologies) on cRGDfK (SEQ ID NO: 2) functionalized coatings on largearea polystyrene dishes (150 cm²) the effectiveness of passaging hMSCswas examined. hMSCs were grown on the coating coupled with cRGDfK (SEQID NO: 2) for 3 days in αMEM with 10% FBS. Versene solution was thenused to successfully passage off the surface and back onto the samesubstrate. Successful reseeding onto the same substrate containing bothadhesive (cRGDfK (SEQ ID NO: 2)) and non-adhesive (blank coating) areas,shows that the substrate was unaltered by the Versene treatment.Further, cell culture with 10% FBS (which contains many proteins andgrowth factors) provides a good test for surface fouling during theprocess.

Versene solution is a phosphate buffered saline (PBS) solutioncontaining 0.48 mM ethenediaminetetraactetic acid (EDTA). EDTA is achemically defined agent used usually in combination with trypsin forhMSC passage. Upon application, EDTA binds the Calcium and Magnesiumions and therefore interferes with the integrin structure, physicallydisrupting the ability of the cell to bind to the peptides on thesurface. However, EDTA has not been shown to be effective for passaginghMSCs on TCPS alone without a protease such as trypsin. Supposedlychemically defined interaction of the hMSCs with the coating allows forpassaging with Versene. The cRGDfK peptide (SEQ ID NO: 2) is known tobind to ανβ3 integrins and potentially ανβ5 and α5β1 integrins as well.(See, Mas-Moruno, C.; Rechenmacher, F.; Kessler, H., Cilengitide: TheFirst Anti-Angiogenic Small Molecule Drug Candidate. Design, Synthesisand Clinical Evaluation. Anti-Cancer Agents in Medicinal Chemistry 2010,10, 753-768 and Shuhendler, A. J.; Prasad, P.; Leung, M.; Rauth, A. M.;DaCosta, R. S.; Wu, X. Y., A Novel Solid Lipid Nanoparticle Formulationfor Active Targeting to Tumor ανβ3 Integrin Receptors Reveals Cyclic RGDas A Double-Edged Sword. Advanced Healthcare Materials 2012, 1,600-608.) It was concluded, therefore, that EDTA was able to disruptthese integrins, allowing for cell detachment. The ability to coat largeplastic dishes, grow hMSCs, and passage them using a chemically definedagent (Versene), makes it feasible to conduct fully chemically definedculture and passaging of hMSCs.

Evaluation of Coating-Peptide Stability in Culture Conditions

An initial goal of the design of VDM containing copolymers was toinvestigate the stability of the peptide-polymer linker. Hence, thestability of the coating-peptide linker (either amide or thioester) wasassessed by soaking experiments in both PBS and αMEM+10% FBS for 2weeks. The average hMSC area on spots patterned with either the adhesivepeptide or the non-adhesive scramble was examined. By first soaking thecoating and then seeding hMSCs, followed by XPS analysis, the followingconclusions regarding the stability of the peptide/polymer link werereached: 1) the amide linkage was stable under both serum and PBSconditions (FIGS. 14 and 15 for cRGDfC (SEQ ID NO: 1) and cRGDfK (SEQ IDNO: 2), respectively); 2) the thioester linkage, though stable in PBS atthe same pH, is labile in serum containing conditions; and 3) thiols aremore efficient in ring opening the azlactone compared to amines, but thethioester bond is more labile under serum conditions. Mechanism of losscould be due to hydrolysis or more likely proteases present in theserum, and/or displacement by primary amines. In fact the labile natureof the thioester bond can be used advantageously to design dynamicallycell responsive surfaces.

Pluripotent Cell Attachment

The utility of this coating is not limited to hMSCs. Pluripotent celltype (H1 embryonic stem cells) were also cultured on the cRGDfK peptide(SEQ ID NO: 2) patterned into 1.1 mm diameter spots. H1s were seededwith ROCK inhibitor (a Rho-associated protein kinase) in E8 media at ahigh density. H1's remained on the surface for 4 days and were confluenton the spots by hour 36. As a control the scramble cRADfK (SEQ ID NO:12) peptide did not show adhesion of H1s, confirming that the cells wereinteracting specifically with the active cRGDfK peptide (SEQ ID NO: 2).H1 pluripotency after culture was not tested in this study, howeverthese initial results demonstrate the broad applicability of thiscoating in other areas of stem cell biology.

CONCLUSIONS

The PEG based copolymer designed herein, provides a well definedtemplate for stem cell culture and passage. The crosslinking of thecoating allows for its use on many different substrate types includingplastic (polystyrene, polycarbonate), silicon, glass and goldsubstrates.

Experimental Design

Materials. Poly(ethylene glycol) methyl ether methacrylate (PEGMEMA,Mn˜300 g/mol), glycidyl methacrylate (GMA), 2-cyano-2-propylbenzodithioate, 2,2′-azobis(2-methylpropionitrile), anisole, acetone,sulfuric acid (H₂SO₄), hydrogen peroxide 30% in water (H₂O₂),cyclopentanone, and ethanol (EtOH) were purchased from Sigma Aldrich Co.2-Vinyl-4,4-dimethyl azlactone (VDM) was a gift from Dr. Steve Heilmannfrom the 3M Corporation. (Milwaukee, Wis.). Silicon wafers (<100>,p-type) were purchase from University Wafer (Boston, Mass.). Humanmesenchymal stem cells (hMSCs) were from Cambrex (North Brunswick,N.J.). Minimum essential medium, alpha (1×; αMEM) was from CellGro(Mannassas, Va.). Trypsin (0.05%) and penicillin/streptomycin were fromHyclone (Logan, Utah). VDM and GMA were purified by vacuum distillation.All other materials were used as received.

Polymer synthesis. Copolymers P(PEGMEMA-r-GMA-r-VDM) were synthesized byRAFT polymerization. In a typical synthesis PEGMEMA (8.3 mmol, 2.49 g),GMA (0.7 mmol, 99.5 mg), VDM (1 mmol, 139.2 mg), are added to a 25 mLSchlenk flask. The solvent anisole (13.6 mL) was added at a monomer tosolvent ratio of 1:5. The chain transfer agent (CTA), 2-cyano-2-propylbenzodithioate (0.01 mmol, 2.2 mg) was added at a total monomer to chaintransfer agent ratio of 1000:1. Last the initiator2,2′-azobis(2-methylpropionitrile), (0.01 mmol, 1.6 mg) was added at ainitiator to CTA ratio of 1:1. The mixture was degassed with threefreeze-pump-thaw cycles. Polymerization was allowed to proceed at 60° C.for 18 hours, after which the mixture was precipitated in n-hexanes andredissolved in tetrahydrofuran three times. The resulting light pinkcopolymer was stored in tetrahydrofuran at −20° C.P(PEGMEMA-r-GMA-r-VDM) was analyzed using gel permeation chromatography(GPC) and proton nuclear magnetic resonance spectroscopy (¹H-NMR).

Substrate preparation. Glass microscope slides purchased from FisherScientific were cut into thirds. Silicon and glass substrates were thensequentially cleaned by sonication in deionized (DI) water, ethanol for5 minutes each and then dried in a stream of air. Following, substrateswere placed in a piranha solution (3:1 H₂SO₄:H₂O₂) at 95° C. for 30minutes, washed with DI water and ethanol, and used within 24 hours ofcleaning. Caution! Piranha reacts violently in contact with organicmatter. Polycarbonate substrates (GraceBio, HybriSlip 22 mm×22 mm),sterile treated tissue culture dishes (Fisher Scientific), and goldsubstrates (EMF, 50 Å Ti, 1000 Å Au) were rinsed with DI water andethanol and used directly.

Film formation and crosslinking. Copolymers were diluted in 100% ethanol(Decon Labs) and spin coated onto the prepared substrates. Theconcentration of the copolymer was 12 mg/mL to achieve thickness of 30nm. Films were promptly annealed for 45 minutes at 160° C. to crosslinkthe film or at 110° C. for 3 hours or 85° C. for 24 hours, all undervacuum.

Coating Stability. Coatings with thicknesses of 30 nm were crosslinkedat 160° C. and put into either deionized (DI) water for 38 days whileincubated at 37° C. Periodic replacement of the aqueous solution wasdone every 3-4 days. Dry coating thickness was determined at designatedtime points by ellipsometry using a Rudolph Auto EL null ellipsometer.Measurements were made at designated time points after drying the filmsunder vacuum overnight using an angle of incidence of 70° andFilmEllipse® software version 1.1 (Scientific Company Intl.).

Contact Angle. Measurements were made on 30 nm coatings crosslinked ontogold, glass, silicon and polycarbonate substrates using a DataphysicsOCA 15 Plus instrument with an automatic liquid dispenser at ambienttemperature. Static water contact angles were measured with a 5 μldroplet of deionized water in 5 different locations on the coatings. Theadvancing and receding contact angles were measured and the data arereported as the average with standard deviation. The same was done foruncoated, bare substrates for comparison.

Elastomeric stencil formation. Elastomeric stencils were created using asoft lithography process already reported in the literature. Briefly, amaster mold was created from SU-8 100 spin coated onto a silicon waferand the pattern was transferred using conventional photolithographytechniques. To prepare the polydimethylsiloxane (PDMS), a 1 to 10 ratioof base to curing agent (w/w) was mixed and degassed for 1 hour. Themixture was cast over the master mold and cured for 6 hours at 85° C.The resulting PDMS template was cleaned by solvent extraction in hexanesovernight.

Peptide Immobilization. Coatings were washed with PBS at pH 7.4 prior topeptide coupling. The cRGDfC peptides (SEQ ID NO: 1) were coupled ateither 0.084 mM, 0.84 mM, or 8.4 mM in PBS at pH 7.4 for 1 hour. ThecRGDfK (SEQ ID NO: 2) peptide was coupled a the same concentrations onlyin a 0.25M sodium phosphate buffer at pH 9.5 with 1.5 M sodium sulfate.For PMIRRAS this pH was adjusted up from pH 7.4 with HCl . The solutionwas bubbled on top of the coating at room temperature and covered toprevent evaporation. This was followed by three 10 minute rinses withdeionized water. For patterning, elastomeric templates with 1.1 mmdiameter spots were placed atop the coating on the desired substrate.The spots were then filled with 1.3 μL, and covered to preventevaporation for 1 hour. After, the solution was aspirated and replacedwith deionized water (1.3 μL) three times. Then the elastomeric templatewas removed, and the coating soaked in PBS for 1 hour. Samples for cellculture were sterilized with 70% ethanol for 20 minutes, transferred toa new sterile 6-well plate, and rinsed three times with sterile PBS toremove ethanol.

PMIRRAS Analysis. Gold substrates (1000 Å, EMF Corporation, TA134)coated with the copolymer were placed at incident angle of 83° in aNicolet Magna-IR 860 Fourier transform IR spectrophotometer equippedwith a photoelastic modulator (PEM-90, Hinds Instruments, Hillsboro,Oreg.), a synchronous sampling demodulator (SSD-100), GWC technologies,Madison Wis.) and a liquid nitrogen cooled mercury-cadmium-telluridedetector. The modulation was set at 1600 cm⁻¹ and 500 scans wereobtained for each sample with a resolution of 8 cm⁻¹. The differentialreflectance IR spectra were then normalized and converted to absorbancespectra using OMNIC software.

XPS Analysis. The measurements were performed with a Thermo ScientificModel K-Alpha XPS instrument using monochromatic Al Kα radiation (1486.7eV). The instrument uses a hemispherical electron energy analyzerequipped with a 128 multi-channel detector system. Survey spectra andhigh-resolution spectra were acquired using analyzer pass energies of200 eV and 50 eV, respectively. The X-ray spot size was 400 μm forsingle point analysis. Depth profiling was done using large Argonclusters with 4000 eV for etching and data was collected every 30seconds using the snap capture function. Data was analyzed usingAvantage XPS software package. Peak fitting was performed usingGaussian/Lorentzian peak shapes and a Shirley/Smart type background. Atleast three points were taken per sample and the averages and standarddeviations are reported.

Cell Culture. The hMSCs (Lonza, Cat PT2501) were expanded at low densityon tissue culture treated polystyrene plates. At passage 6 the cellswere harvested using a 0.05% trypsin solution and suspended in 1 mL ofαMEM. In a rectangular 6-well plate, 4 mL of αMEM containing 1%penicillin/streptomycin and 10% MSC qualified fetal bovine serum (FBS)(HyClone Cat # SH30070) was added. hMSCs were seeded at 5,000 cells percm² into each well for patterned peptide spots and 7,000 cells per cm²for large area coatings. Wells were then gently rocked to evenlydistribute the cells. Cells were incubated at 37° C. and 5% CO₂ topromote cell attachment for 6 hrs. At the end of the attachment period,the medium was aspirated from the wells and gently washed with sterile1×PBS (pH 7.4) to remove any dead or loosely attached cells, after whichcells were fixed with 3.7% formaldehyde in PBS buffer for 15 minutes.

Immunohistochemistry. Staining of the actin cytoskeleton and nuclei wasperformed as directed by the manufacturer (catalogue no. FAK100,Millipore, Mass.). Substrates were then washed with a solutioncontaining 0.05% Tween 20 in a 1×PBS solution. Cells were permeabilizedusing 0.1% Triton X-100 in 1×PBS for 5 min. The mats were rinsed twicewith a wash buffer and then blocked to prevent nonspecific antibodyadsorption using 1% BSA in 1×PBS for 30 min. The mats were thenincubated in 1×PBS with the TRITC-conjugated phalloidin for 1 h. Afterrinsing with the wash buffer three times, mats were dipped face downonto a glass slide with a drop of Prolong Gold anti-fade mounting mediawith DAPI (Invitrogen). Cells were imaged on an inverted microscopeequipped with FITC, TRITC, and DAPI filter cube sets. For thisexperiment 3 replicates of each condition were used.

Cell culture stability assay. Coatings patterned with cRGDfK (SEQ ID NO:2) or cRGDfC (SEQ ID NO: 1) or the non-adhesive cRADfC (SEQ ID NO: 13)or cRADfK (SEQ ID NO: 12) on 2 mm diameter spots were sterilized byincubating in 70% ethanol for 20 minutes, which was followed by tworinses with sterile PBS. Coatings were then incubated at 37° C. inαMEM+10% FBS or PBS for 2 weeks. Media was replaced every 3-4 days.After 2 weeks, the samples were rinsed with sterile PBS and deionizedwater, then hMSCs were seeded at 10,000 cells/cm² and let adhere for 6-7hours before fixing and staining for actin and nuclei.

Passage of hMSCs. Plastic polystyrene dishes (round, Fisher Scientific)with area of 150 cm² or 70 cm² were coated with a 30 nm polymer coating,as previously described, and crosslinked at 85° C. The cRGDfK peptide(SEQ ID NO: 2) was coupled to approximately half of the dish, using aPDMS strip as a divider. The peptide was coupled at 0.084 mMconcentration in the 1.5 M Sodium Sulfate buffer at pH 9.5 for 1 hour atroom temperature. The dish was then soaked in PBS for 30 minutes,followed by 70% ethanol for 20 minutes to sterilize. hMSCs (P6) wereseeded at low density 1,000 cells/cm² in αMEM+10% FBS. Cells reachedconfluence in 3 days. Versene solution (Life Technologies) was warmed to37° C. in a water bath. Media above the cells was aspirated and rinsedtwice with PBS before placing 10 mL of Versene solution into the dishfor 1.5 minutes. Versene was then aspirated and fresh αMEM (5 mL threetimes) was used to remove the cells from the surface. On the samesurface, 20% of the cells were reseeded down and imaged after 24 hours.

H1 pluripotent stem cell culture. H1 human ESCs (WiCell) were maintainedon 6-well plates coated with Matrigel (8.7 μg/cm²; BD Biosciences) inEssential 8 medium (E8; Invitrogen) with daily media exchange, andpassaged using Versene (Invitrogen) every 3 to 4 days. For substrateseeding studies, cells were washed with PBS and incubated with TrypLE(Invitrogen) at 37° C. for 5 minutes to promote singularization. Cellsuspensions were diluted with E8 supplemented with 5 μM Rho kinaseinhibitor (Y-27632; CalBiochem) and pelleted by centrifugation at 200 gfor 5 minutes before counting by hemacytometer and seeding at 1.8×10⁵cells/cm².

Statistical Analysis. Values reported are the mean plus or minus thestandard deviation. Experiments were analyzed using a two-tailedStudent's t-test. Data were considered “statistically significant” ifp<0.05.

Example 2

This example illustrates a P(VDM-r-GMA) polymer coating thatincorporates VDM chemistry for use as a template for stem cell growthand expansion.

Copolymers VDM-r-GMA were synthesized by atomic transfer radicalpolymerization (ATRP) with a 200 to 1 monomer to initiator ratio.Briefly, VDM (9.0 mmol, 2.5 g) and GMA (0.5 mmol, 0.28 g) were added toa Schlenk flask with copper (I) bromide (0.05 mmol, 21.4 mg),N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA, 0.05 mmol, 26 mg)and 3 mL toluene. The flask was degassed by three freeze-pump-thawcycles. The flask was then heated to 60° C. in an oil bath, and theinitiator ethyl-2-bromoisobyrate (0.05 mmol, 29.3 mg) was injected tostart the reaction. The reaction was allowed to proceed for 4.5 hours,after which the copolymer was precipitated in hexane and dried. H¹ NMRcharacterization showed the composition to be 11.9 mol. % GMA and 88.1mol. % VDM. Gel permeation chromatography gave a number averagemolecular weight of 10,815 Da and a dispersity of 1.2. The VDM-r-GMAcopolymer is not soluble in water or ethanol mixtures, limiting itsapplicability to plastic substrates. Therefore, the copolymer was spincoated from cyclopentanone (12 mg/ml) onto glass substrates andcrosslinked at 160° C. for 6 hours. The copolymer VDM-r-GMA was modifiedwith functional amines after crosslinking. Unmodified films promotedadhesion and spreading of hMSCs in cell culture medium with 10% fetalbovine serum. To inhibit adhesion, a 660 μM solution ofmethoxypolyethylene glycol amine 750 (Sigma Aldrich) in deionized waterwas applied for 1 hour. hMSCs were seeded at 5,000 cells/cm' at passage7 and allowed to adhere for 6 hours, after which they were imaged.Unmodified (blank) areas of the coating readily promoted cell adhesion,which was non-specific in nature. The region that was modified withPEG-amine resisted cell adhesion.

The word “illustrative” is used herein to mean serving as an example,instance, or illustration. Any aspect or design described herein as“illustrative” is not necessarily to be construed as preferred oradvantageous over other aspects or designs. Further, for the purposes ofthis disclosure and unless otherwise specified, “a” or “an” means “oneor more.”

The foregoing description of illustrative embodiments of the inventionhas been presented for purposes of illustration and of description. Itis not intended to be exhaustive or to limit the invention to theprecise form disclosed, and modifications and variations are possible inlight of the above teachings or may be acquired from practice of theinvention. The embodiments were chosen and described in order to explainthe principles of the invention and as practical applications of theinvention to enable one skilled in the art to utilize the invention invarious embodiments and with various modifications as suited to theparticular use contemplated. It is intended that the scope of theinvention be defined by the claims appended hereto and theirequivalents.

What is claimed is:
 1. A cell culture substrate comprising: a substratehaving a surface; and a film comprising crosslinked random copolymers onthe surface of the substrate, the crosslinked random copolymers havingbackbone chains comprising polymerized monomers comprising covalentlylinked peptide chains and monomers that provide covalent crosslinksbetween the backbone chains, the crosslinked random copolymerscomprising the structure:

wherein x any y represent the mole fractions of the crosslinked monomersand the monomers comprising covalently linked peptide chains;

represents a crosslink to another copolymer backbone chain; Peprepresents a peptide chain; and the crosslinks and peptide chains aredistributed randomly along the copolymer backbone.
 2. The cell culturesubstrate of claim 1, wherein the copolymer comprises from about 1 toabout 15 mole percent of the monomers that provide covalent crosslinksbetween the backbone chains and from about 99 to about 85 mole percentof the polymerized monomers comprising covalently linked peptide chains.3. The cell culture substrate of claim 1, wherein the backbone chains ofthe random copolymers further comprise polymerized monomers comprisingcovalently linked polyethylene glycol chains, the crosslinked randomcopolymers comprising the structure:

wherein x, y and z represent the mole fractions of the crosslinkedmonomer, the monomers comprising covalently linked peptide chains andthe monomers comprising covalently linked polyethylene glycol chains; nrepresents the number of repeat units in the polyethylene glycol chain;

represents a crosslink to another copolymer backbone chain; Peprepresents a peptide chain; and the crosslinks, the peptide chains andthe polyethylene glycol chains are distributed randomly along thecopolymer backbone.
 4. The cell culture substrate of claim 1, whereinthe film has a thickness no greater than about 30 nm.
 5. The cellculture substrate of claim 1, wherein the substrate is a polymericsubstrate.
 6. The cell culture substrate of claim 4, wherein the film isnot covalently bound to the substrate.
 7. A method of culturing stemcells using the cell culture substrate of claim 1, the method comprisingseeding the stem cells onto the cell culture substrate and culturing theseeded stem cells in a cell culture medium under cell culturingconditions.
 8. A coated substrate comprising: a substrate having asurface; and a film of crosslinked random copolymers on the surface ofthe substrate, the crosslinked random copolymers having backbone chainscomprising polymerized 4,4-dimethyl-2-vinylazlactone monomers,polyethylene glycol methyl ether methacrylate monomer and monomers thatprovide covalent crosslinks between the backbone chains, the crosslinkedrandom copolymers comprising the structure:

wherein x, y and z represent the mole fractions of the crosslinkedmonomer, the 4,4-dimethyl-2-vinylazlactone monomer and the polyethyleneglycol methyl ether methacrylate monomer; n represents the number ofrepeat units in the polyethylene glycol chain;

represents a crosslink to another copolymer backbone chain; and thecrosslinks, the azlactone groups and the polyethylene glycol groups aredistributed randomly along the copolymer backbone.
 9. The coatedsubstrate of claim 8, wherein the random copolymer comprises from about1 to about 15 mole percent of the polymerized monomers that providecovalent crosslinks between the backbone chains, from about 15 to about60 mole percent of the polymerized 4,4-dimethyl-2-vinylazlactonemonomer, from about 30 to about 85 mole percent of the polymerizedpolyethylene glycol methyl ether methacrylate monomer, and no greaterthan about 30 mole percent of additional monomer.
 10. A cell culturesubstrate comprising: a substrate having a surface; and a filmcomprising crosslinked random copolymers on the surface of thesubstrate, the crosslinked random copolymers having backbone chainscomprising polymerized polyethylene glycol methyl ether methacrylatemonomers, monomers comprising covalently linked peptide chains, andmonomers that provide covalent crosslinks between the backbone chains,the crosslinked random copolymers comprising the structure:

wherein x, y and z represent the mole fractions of the crosslinkedmonomer, the monomers comprising covalently linked peptide chains andpolyethylene glycol methyl ether methacrylate monomers; n represents thenumber of repeat units in the polyethylene glycol chain;

represents a crosslink to another copolymer backbone chain; Peprepresents a peptide chain; and the crosslinks, the peptide chains andthe polyethylene glycol groups are distributed randomly along thecopolymer backbone.
 11. The cell culture substrate of claim 10, whereinthe random copolymer comprises from about 1 to about 15 mole percent ofthe polymerized monomers that provide covalent crosslinks between thebackbone chains, from about 15 to about 60 mole percent of thepolymerized monomers comprising covalently linked peptide chains, fromabout 30 to about 85 mole percent of the polymerized polyethylene glycolmethyl ether methacrylate monomer, and no greater than about 30 molepercent of additional monomer.
 12. A method of culturing stem cellsusing the cell culture substrate of claim 10, the method comprisingseeding the stem cells onto the cell culture substrate and culturing theseeded stem cells in a cell culture medium under cell culturingconditions.